(PI: Gonzalo E. Torres, Neurobiology, Pitt). The PI has identified the synaptic vesicle protein synaptogyrin-3 (SG3) as a DAT-interacting protein. Based on preliminary data, a central hypothesis is that the DAT/SG3 interaction influences DAT function, and ultimately dopamine homeostasis. This may be due to a link between plasma membrane DAT-mediated uptake and the vesicular dopamine storage system, or alternatively, through regulation of DAT targeting, recycling, and/or intrinsic transporter activity. The project combines biochemical, molecular, and functional approaches in vitro, and in vivo with cells in culture, synaptosomal preparations, purified synaptic vesicles, and whole animals, to generate a detailed structural, functional, and subcellular characterization of the DAT/SG3 interaction. Specifically, the aims are to identify the residues involved in the DAT/SG3 interaction and examine their interaction’s specificity; and investigate SG3’s impact on DAT function, the regulation of the DAT/SG3 interaction, and the subcellular location for DAT/SG3 interaction.

The long-term goal of this project is to understand the mechanisms involved in the regulation of dopamine homeostasis by DAT, and how these mechanisms are altered by psychostimulants. We note the close connection between the goals and interests of FRP4, FRP5, and FRP6, and naturally, the computational methods that will be extended/adapted to DA by the Center will serve all three FRPs. We anticipate our Center will facilitate cross-interactions among the FRPs, as well as help increase the effectiveness of the research ongoing in each. The discovery of novel DAT interacting proteins may suggest novel mechanisms associated with DAT’s activity; in turn, such mechanisms will have an important impact in the regulation of dopamine homeostasis. Core B has expertise in modeling the protein/protein interactions, in conjunction with the Target/off-target and polydrug addiction algorithms developed by Core A, which will provide in-depth information for better understanding SG3/DAT interaction in aid of new drug design.

Using quantitative live-cell fluorescence microscopy (Sorkin lab; U PITT) and molecular modeling (Bahar lab), they investigated the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-64 on the plasma membrane distribution of wild-type DAT and two DAT mutants. Read more

Core C proposes a new approach, Linear Regression with Validated Association (LRVA) that discovers variants by utilizing the knowledge of known associations, making the variants with weaker signals easier to be identified. They have applied their method to Drug Abuse Disorders data set collected from CEDAR provided to them by Dr. Michael Vanyukov (PI of a FRP), and discovered nearly two dozen novel SNPs associated with drug abuse disorders. Literature search suggests that many of these SNPs reside in genes previously known to be involved with substance abuse. Read more